In the first period for which this grant has been funded we demonstrated that one response of tumor cells to toxic levels of exposure to a wide array of anticancer drugs is hypermethylation of their genomes. We also demonstrated that random gene silencing that occurs as part of this phenomenon will occasionally transcriptionally inactivate gene loci whose products are required for the activation of commonly used drugs to their cytotoxic forms. We have shown that this seeds the tumor cell population with subclones of cells expressing resistance to these drugs. We call the cells comprising such subclones "epimutants" since their phenotype is induced by nonmutational means and is therefore potentially reversible. Drug-induced DNA hypermethylation thus represents an interesting adaptive response of tumor cells to toxic concentrations of anticancer drugs. In this application for renewal, we propose to extend our previous in vitro studies to clinically applicable in vivo model systems. These model systems include (1) HT-29SFm- human colonic adenocarcinoma cells growing in nude mice and investigated for hypermethylation-mediated silencing of the thymidine kinase gene causing resistance to 5-fluorodeoxyuridine; and (2) MOLT-4m-human T-cell leukemia cells grown in nude mice and investigated for hypermethylation-mediated silencing of the HPRT gene causing resistance to 6-thioguanine; hypermethylation-mediated silencing of the deoxycytidine kinase gene causing resistance to cytosine arabinoside; and hypermethylation-mediated silencing of the thymidine kinase gene causing resistance to AZT. Since we have shown in our in vitro work that drug resistance resulting from such drug-induced DNA hypermethylation can be inhibited and even reversed by the appropriate application of methylation inhibitors, our currently proposed studies have the potential for significant clinical importance.